PROJECT SUMMARY Strict glycemic control to maintain normal blood glucose levels is crucial to the health of more than 23 million diabetic patients in the United States. However, up to two-thirds of these patients do not monitor their glucose levels regularly because of the pain and inconvenience of current blood glucose detection. Ideally, individuals could detect glucose levels from noninvasive samples such as exhaled breath condensates (EBC). Because glucose is 2500x less concentrated in breath than in blood, these measurements require highly sensitive and specific biosensors that can rapidly detect glucose concentration changes at the M level. Currently, even the most exquisitely sensitive biosensors require large samples volumes and/or long detection times to measure changes in glucose concentration at these levels. To address this gap, the goal of this project is to develop and validate a highly sensitive and selective glucose biosensor inspired by the high affinity binding of glucose to the E. coli glucose/galactose binding protein (GBP). Engineered GBP will be covalently bound to fluorescent quantum dots (QDs) directly from cell lysate mixtures with a chemoenzmatic tag. This site selective tagging chemistry negates the need for purification step that may negatively affect protein functionality. In addition, a homogeneous population of GBP is presented on the quantum dot surface that is expected to maximize glucose binding affinity. QD fluorescence is initially quenched by binding of galactose- bound fluorophore. When samples containing glucose are introduced, the galactose-quencher is displaced by the glucose and luminescence of the QD increases proportionally. The sensitivity, specificity, precision, accuracy, repeatability, and resolution of the biosensor's dose-response to glucose in simulated breath condensate samples will be characterized via fluorometric assays and high-performance anion exchange chromatography with pulsed amperometric detection, the gold standard for carbohydrate characterization. Finally, the GBP-QD biosensor will be subjected to mock breath condensate solutions simulating diabetic and lung pathologies with altered pH, electrolytes, ketones and acetone. Ultimately, this biosensor will enable noninvasive blood glucose measurements that provide patients and caregivers with a rapid and pain-free method to monitor blood glucose.